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Anatomy of an engineered NAD-binding site
P R Mittl1, A Berry, N S Scrutton
1Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany.
Protein Science : a Publication of the Protein Society
|September 1, 1994
Summary
Researchers engineered Escherichia coli glutathione reductase to use NAD instead of NADP by introducing specific mutations. Structural analysis revealed subtle changes explaining the altered coenzyme specificity and long-range effects of mutations.
Area of Science:
- Biochemistry
- Structural Biology
- Enzyme Engineering
Background:
- Escherichia coli glutathione reductase naturally utilizes NADP+ as a coenzyme.
- Modifying enzyme specificity is crucial for understanding protein function and engineering new biocatalysts.
Purpose of the Study:
- To investigate the structural basis for switching the coenzyme specificity of E. coli glutathione reductase from NADP+ to NAD+.
- To analyze the structural consequences of point mutations designed to alter coenzyme binding.
Main Methods:
- Site-directed mutagenesis to create an NAD-dependent glutathione reductase mutant.
- High-resolution X-ray crystallography to determine structures of wild-type and mutant enzymes, both apo and ligated with NADP+ or NAD+.
- Structural comparison and analysis of subtle differences in enzyme conformation and binding sites.
Main Results:
- A set of seven point mutations successfully switched the coenzyme specificity from NADP+ to NAD+.
- Crystal structures revealed subtle conformational changes, including peptide rotation and altered ribose pucker, near the 2'-phosphate binding site.
- The engineered NAD-binding site exhibited a less rigid structure compared to the wild-type NADP+ site.
- A displacement of NAD versus NADP+ in the bound state elongated the electron transfer pathway, potentially explaining reduced catalytic efficiency.
Conclusions:
- Specific point mutations can effectively alter enzyme coenzyme specificity by inducing subtle structural changes.
- The study highlights the complex, long-range effects of mutations on enzyme structure and function, even when mutations are distant from the active site.
- Understanding these structural rearrangements provides insights into enzyme-coenzyme interactions and guides future enzyme engineering efforts.